Abstract

Gram-negative bacteria produce outer membrane vesicles (OMVs) that serve a variety of functions related to survival and pathogenicity. Periplasmic and outer membrane proteins are naturally captured during vesicle formation. This property has been exploited as a method to derive immunogenic vesicle preparations for use as vaccines. In this work, we constructed a Salmonella enterica serovar Typhimurium strain that synthesized a derivative of the pneumococcal protein PspA engineered to be secreted into the periplasmic space. Vesicles isolated from this strain contained PspA in the lumen. Mice intranasally immunized with the vesicle preparation developed serum antibody responses against vesicle components that included PspA and Salmonella-derived lipopolysaccharide and outer membrane proteins, while no detectable responses developed in mice immunized with an equivalent dose of purified PspA. Mucosal IgA responses developed against the Salmonella components, while the response to PspA was less apparent in most mice. Mice immunized with the vesicle preparation were completely protected against a 10× 50% lethal dose (LD₅₀) challenge of Streptococcus pneumoniae and significantly protected against a 200× LD₅₀ challenge, while control mice immunized with purified PspA or empty vesicles were not protected. These results establish that vesicles can be used to mucosally deliver an antigen from a Gram-positive organism and induce a protective immune response.

PspA is associated with the lumens of OMVs. Purified S. enterica serovar Typhimurium vesicles (OMV-PspA) were incubated with proteinase K (PK) and without proteinase K for 30 min at 37°C. Some vesicle preparations were lysed with 1% SDS, in the absence or presence of PMSF, prior to treatment with proteinase K. After proteinase K treatment, vesicles were subjected to SDS-PAGE and immunoblot analysis using anti-PspA antibodies. The leftmost lane contains molecular mass markers.

Serum IgG responses in mice immunized with PspA, OMVs, or OMVs containing PspA (OMV-PspA). Groups of mice were immunized intranasally four times at weekly intervals with 350 ng PspA, 10 μg PspA, OMVs, OMV-PspA (an amount equivalent to 350 ng PspA), or PBS. OMVs and OMV-PspA were isolated from strain χ9281(pYA3493) and χ9281(pYA4088), respectively. Sera were collected 3 weeks after the final immunization. Sera were diluted 1:100 and analyzed by ELISAs for IgG responses against OMV (A), S. Typhimurium LPS (B) Salmonella OMPs (SOMPs) (C), and PspA (D). Combined data from three independent experiments are shown. The means ± standard errors (error bars) for the groups from triplicate experiments are shown. Each symbol represents the value for an individual mouse. IgG responses in mice immunized with OMV or OMV-PspA were significantly different from the responses of the control mice given PBS and other groups given OMVs, LPS, and SOMPs (*, P < 0.05). The anti-PspA responses in mice immunized with OMV-PspA were significantly different (P < 0.05) from those of all other groups (*).

Secretory IgA responses to OMVs in vaginal secretions. Vaginal secretions were collected from immunized mice 3 weeks after the final immunization, and the mucosal IgA responses against OMV(A) and PspA (B) were determined by ELISAs. The vaginal lavage fluid samples were diluted 1:10 and assayed. Combined data from three independent experiments are shown. The means ± standard errors (error bars) for the groups from triplicate experiments are shown. Each symbol represents the value for an individual mouse. OMV-specific IgA responses in OMV- and OMV-PspA-immunized groups were significantly different from the control group given PBS (*, P < 0.05). There were no differences in the anti-PspA responses between groups.

Protective immunity against S. pneumoniae WU2 challenge. Groups of mice were immunized intranasally four times at weekly intervals with 350 ng PspA, 10 μg PspA, OMVs, OMVs containing PspA (OMV_PspA) (an amount equivalent to 350 ng PspA), or PBS. Five weeks after the final immunization, all mice were challenged with either 2,000 CFU (A) or 40,000 CFU (B) of S. pneumoniae WU2. Mortality was monitored for 2 weeks after the challenge. The values for the OMV_PspA groups were significantly different from those for the control groups given PBS (*, P < 0.05; **, P < 0.0001).